CHM 3240 – Biochemistry

Polymers of Amino AcidsPROTEINS

Protein sequenceYMGCFTSSGLIVVEHY...

Structure

Function

DNA (gene)

mRNA

Amino Acid H |H2N - Ca - COOH | R

Protein = Polymers of Amino Acids

Carboxyl groupamino group

a-carbon

Side chain

If R = -CH3 (ALA), is Ca chiral?

a) yes b) no

All amino acids are chiral (except glycine)Proteins only contain L amino acids These are all S except for Cys which is R

Sidechain Categories

Nonpolar: hydrocarbon + Met (C-S-C)

Acidic: Ocarboxylic Acid ||

- C - OH

Basic: Amine (-NH2)

Polar: hydroxyl (OH) Cys (S-H)

Oamide ||

- C – NH2

Nonpolar R Groups

These amino acids side chains can form hydrogen bonds.Cysteine can form disulfide bonds.

Acidic Amino AcidsShown in ionized form – dominant at pH 7.4

Basic Amino AcidsShown in dominant form at pH 7.4

These amino acid side chains absorb UV light at 270–280 nm

nonpolar polar Polar with some NP

character

What is Beer’s Law?

a) The MW of a molecule is proportional to its volume.

b) The Pressure of a gas is proportional to the # of gas molecules. (cst V)

c) The absorbance of a molecule is proportional to its concentration.

d) The solubility of a molecule in water is proportional to its dipole moment.

Proteins are typically detected by their absorbance (A280). Beer’s Law: A = ebC applies. e is determined mostly by the # of Trp (& Tyr) residues in the protein.

What is Beer’s Law?

e280 = 5580 M-1 cm-1

e275 = 1405 M-1 cm-1

e257 = 195 M-1 cm-1

Essential − Required in your dietary proteinCan’t be synthesized from other dietary sources(in bolded print on amino acid handout)

Nonessential − Not required in diet Can be made from carbohydrates + N from other amino acids.

The RDI (recommended daily intake) of protein (~ 60 g), requires that all essential amino acids are represented in significant amounts. (0.66 g/kg)

Amino Acid – acid/base properties

H |H2N - Ca - COOH | CH3

-COOH ↔ -COO- + H+

acid base

Ka = [-COO-] [H+] [-COOH]

-NH2 + H+ ↔ -NH3+

base acid

Ka = [-NH2] [H+] [-NH3

+]

pKa = - log Ka

Write an equation showing –COOH acting as an acid.Write the equation representing Ka for this reaction.

Amino Acid (Ala) – acid/base properties

H |H2N - Ca - COOH | CH3

pK = 2.3 see column pK carboxy on aa handout

pK = 9.7see column pK aminoon aa handout

― +H3N

Both groups > 90% ionized between ~ pH 3 – 8.5

pH = pK + log (b/a)Henderson Hasselbach Equation

Acidic and basic side chains may also be ionized with the fraction ionization determined by the HH equation. (see column pK side chain on aa handout)

pH = pK + log (b/a)

Low pH Neutral pH High pH

pK ~ 2 pK ~ 9

Non-ionizable side chain

Side Chain ionization Aspartic acid – pK = 4.1What fraction is in acid vs. base form at pH = 5.0

O ||— C — OH ↔ acid (a) base (b)

O ||— C — O- + H+

pH = pK + log (b/a)

5.0 = 4.1 + log (b/a)log (b/a) = 5.0 – 4.1 = 0.9b/a = 10log (b/a) = 100.9 = 7.9 or 7.9:1

Fraction base = 7.9/(1+7.9) = 0.89 or 89% base

89% base11% acid

Side Chain ionization Aspartic acid – pK = 4.1What fraction is in acid form at pH = 3.6 a) 0.50 b) 0.24 c) 0.32 d) 0.76

O ||— C — OH ↔ acid (a) base (b)

O ||— C — O- + H+

pH = pK + log (b/a)

3.6 = 4.1 + log (b/a)log (b/a) = 3.6 – 4.1 = -0.5

b/a = 10log (b/a) = 10-0.5 = 0.32 or 0.32:1Fraction base = 0.32/(1.32) = 0.24 or 24% base

24% base76% acid

pH - pK ~ frac base

~ frac acid

+3 0.999 0.001+2 0.99 0.01+1 0.90 0.100 0.50 0.50-1 0.10 0.90-2 0.01 0.99-3 0.001 0.999

Qualitative Estimate Henderson Hasselbach EquationEach pH unit variation from pK shifts equilibrium 10x

Peptide Bond

O H H ||H-N - C - C | R1

O H H || -N - C - C - OH | R2

- OH H

O H H ||H-N - C - C | R1

O H H || -N - C - C - OH | R2

+ HOH

Dipeptide

POLYPEPTIDE CHAIN

O O O O H || H H || H H || H H || _H3N-C-C-N-C-C-N-C-C-N-C-C-O | | | | R1 R2 R3 R4

+

AMINO END

CARBOXY END

Sequence (primary structure) = R1-R2-R3-R4 …. Etc.

Sequence = YGGFLListed order of aas from amino end to carboxyl end

Note bond angles make extended backbone ‘zig-zag’ and side chains alternate sides in extended chain

Peptide ― A polymer composed of a small number of amino acids. dipeptide, tripeptide, oligopeptide, polypeptide

Peptides lack (too small) a specific folded structure.

Glutathione (ROS protection) = gGlu-Cys-Gly

Leu Enkephalin (neurotransmitter) = YGGFL

Aspartame (artificial sweetener) = Asp-Phe-O-CH3

Peptide examples

Protein ― A molecule composed of 1 or more folded polypeptide chains that performs a biochemical function.

Myoglobin sequence 153 aa; 17053 MW; GLSDGEWQLV LNVWGKVEAD IPGHGQEVLI RLFKGHPETL EKFDKFKHLK SEDEMKASED LKKHGATVLT ALGGILKKKG HHEAEIKPLA QSHATKHKIP VKYLEFISEC IIQVLQSKHP GDFGADAQGA MNKALELFRK DMASNYKELG FQG

Protein = Polymers of Amino Acids

pI = 7.3

e280 = 13,980 M-1 cm-1.

Review

a-amino acid structure amino group, carboxyl group, H atom, side chain optical isomerism – nature makes only L-amino acids

amino acid classification nonpolar – C & H only + methionine polar – alcohol/amide + cysteine + Tryptophan acidic - carboxyl group basic – amino group (not aromatic)

aromatic – absorb near UV light A280 used to measure [proteins]

Key questions about Proteins

What is its sequence and amino acid composition?What is its three-dimensional structure?

(x-ray crystallography or 2D NMR)

What is its function? What does it do?What is its mechanism of action? How does it work?

Is its function regulated? If so, how?Is it related to other proteins?

Where is it localized within the cell/body?

What are its physical properties? MW, pI, e280, etc.

The methods part of Chapter 3 will be covered (in part) after Chapters 4 and 5

Studying Proteins requires separation

• Separation relies on differences in physical and chemical properties– Charge (+ if pH < pI - if pH > pI)

– Size (MW 1 kiloDalton = 1000 g/mol)

– Affinity for a ligand (related to function – what does it bind?)

– Solubility - (NH4)2SO4 and salting out• Chromatography is commonly used for preparative

separation

CHROMATOGRAPHY

Mobile Phase — solvent + solutes to be separated.

solutes have differential adherence to matrix.

Stationary Phase — solid support matrix

Chromatography Types Adsorption (nonspecific VdW)

Gel Filtration (size exclusion)

Ion Exchange (charge)

Affinity (specific ligand)

Protein ― A molecule composed of 1 or more folded polypeptide chains that performs a biochemical function.

Myoglobin sequence 153 aa; 17053 MW; GLSDGEWQLV LNVWGKVEAD IPGHGQEVLI RLFKGHPETL EKFDKFKHLK SEDEMKASED LKKHGATVLT ALGGILKKKG HHEAEIKPLA QSHATKHKIP VKYLEFISEC IIQVLQSKHP GDFGADAQGA MNKALELFRK DMASNYKELG FQG

Protein = Polymers of Amino Acids

pI = 7.3

e280 = 13,980 M-1 cm-1.

Separation relies on differences in physical and chemical properties.Myoglobin is found in muscle cells.

Column Chromatography

A has strongest adherence to matrix

C bonds weakly to Matrix and elutes 1st

Separation by Size

0

0.2

0.4

0.6

0.8

1

1.2

0 10 20 30 40 50 60 70

Large MW standard (blue dextran) = void volume

Smaller proteins

Largerproteins

A280

Fraction #

Separation by Charge

If pH > pI then the protein hasa negative charge (-).

If pH = pI then the protein hasNo net charge, such that the # of(+) groups = # (-) groups.

If pH < pI then the protein hasa positive charge (+).

Polymerized (and cross-linked) acrylamide is used for protein electrophoresis (and DNA sequencing)

PAGE = Polyacrylamide Gel Electrophoresis

Apply protein mixture to wells.

Apply voltage to system.

(-) proteins migrate toward (+) electrode.

Different proteins migrate at different rates dependent on charge:size ratio.

v = Ez/f

Types of PAGE

Isoelectric charge pIfocusing

Type Separation Determines

standard charge “pattern” size

SDS size MW

v = Ez/fV = migration rateE = voltage applied (same for all proteins in mixture)Z = protein charge (related to pH and pI)F = frictional coefficient (related to protein size and gel density (constant).

SDS PAGE: Molecular Weight

• SDS – sodium dodecyl sulfate – a detergent

• SDS micelles bind to and unfold all the proteins– SDS gives all proteins an uniformly negative charge with similar shape.– Rate of movement will only depend on size: small

proteins will move faster.

SDS PAGE: Separates proteins based on size.v = Ez/f all proteins are uniformly (-) but larger proteins have large “f” frictional component which slows down their migration.

MW ~ 40,000

Which functional group is not part of polar side chains.

A. -COOH O ||

B. – C – NH2

C. -OH

Review Questions – amino acids

All natural amino acids in proteins are? A. nonpolar

B. L stereoisomersC. S stereoisomersD. g-amino acids

The Val side chain …. -CH2 – CH3 is ……? | CH3

A. Nonpolar B. Polar C. Acidic D. Basic